Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
J Am Chem Soc. 2012 Jul 18;134(28):11525-32. doi: 10.1021/ja3020555. Epub 2012 Jul 5.
A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, "flow-and-stop" control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.
一种方法被开发出来以检测在毛细管细胞中自由扩散的单分子的荧光强度信号。一个基于球形反射镜的独特光学系统被设计出来以实现对荧光强度的定量检测。此外，“流动-停止”控制样品可以将单分子的观察时间延长到几秒钟，比使用典型的共焦方法可获得的观察时间长 1000 多倍。我们使用该方法仔细研究了标记细胞色素 c 在去折叠状态下的荧光时间序列。基于局部平衡态分析的轨迹时间序列分析揭示了毫秒时间尺度上动态不同的亚稳态。该系统为蛋白质折叠能景观的实验特性研究提供了新的途径。
